Regeneration and development. An amphibian call to arms.

BACKGROUND: Unlike axolotls, the urodele Notophthalmus viridescens completes two metamorphoses and emerges from its aquatic environment to mate as a fully terrestrial adult. Larval and adult limb regeneration are commonly treated as roughly equivalent processes and, at least in part, as a recapitulation of embryonic development. RESULTS: We compared larval limb development to regeneration of both larval and adult forelimbs and found that there are substantial differences in developmental pattern among larvae and adults. The larval pattern of preaxial dominance is absent in adult regenerates: adult regenerates instead develop digits synchronously, and they do so before proximal autopodial elements have formed discrete aggregation zones. By contrast, larval regenerates follow a pattern of sequential digit formation from anterior to posterior, like their embryonic limb buds. CONCLUSIONS: Based upon these morphological clues, we conclude that larval regenerates are unlikely to exhibit features of epimorphic regeneration seen in adults, but are more likely to represent a form of developmental regulation. Furthermore, we confirm that post-metamorphic limb regeneration is not a simple recapitulation of ontology at the morphological level. These distinctions may help to explain and interpret some experiments and observations of regeneration in neotenic or paedomorphic urodeles.

Revisited - Failure of tetrodotoxin to protect red-spotted newts, Notophthalmus viridescens, from endoparasites.

Red-spotted newts, Notophthalmus viridescens, contain tetrodotoxin (TTX) and its analogue 6-epiTTX in variable concentrations. In a follow-up study, newts were sampled from a pond in Pennsylvania, USA, in 2010, 2014, and 2018. Their toxin levels were assayed by liquid-chromatography-fluorescence detection (LC-FLD), and assessment of their infection with endoparasites such as nematodes and helminths was performed by histological examination of internal organs. In the 2010 and 2014 samples, average prevalence of parasite infection was 53 and 60%, respectively, but reached 100% in the 2018 sample, where metacercaria stages of the digenean trematode genus Australapatemon/Apatemon (family: Strigeidae) were predominant causing severe tissue damage in liver and kidney. Mean values of TTX and 6-epiTTX were not significantly different in parasitized or parasite-free newts over the study period, confirming previous findings that host toxicity and parasite load are not negatively correlated. Whereas the role of TTX in defence against predators is undisputed, its efficacy to prevent parasitic infections is less obvious. Toxin-resistance of various metazoan parasites may promote their widespread occurrence in poisonous newts.

Differential plasticity of membrane fatty acids in northern and southern populations of the eastern newt (Notophthalmus viridescens).

Seasonal changes in membrane composition and metabolic activity allow many temperate ectotherms to contend with changes in body temperature, but few studies have investigated whether the plasticity of these traits has diverged within a single species. Therefore, we studied the effects of thermal acclimation on the membrane fatty acid composition and the activities of cytochrome c oxidase (CCO) and citrate synthase (CS) in the skeletal muscle and liver of eastern newts from Maine and Florida. Newts were acclimated to either 6 °C or 28 °C for 12 weeks prior to experiments. Cold acclimation resulted in a lower saturated fatty acid (SFA) content in the muscle membranes of both populations. SFA content in liver was lower in cold compared to warm-acclimated newts from Florida, but acclimation did not affect SFA content in liver membranes of the Maine population. In liver, cold acclimation resulted in a higher monounsaturated fatty acid (MUFA) content in the Florida population and a higher polyunsaturated fatty acid (PUFA) content in the Maine population. Regardless of acclimation conditions, the muscle and liver membranes of the Maine population had higher SFA and PUFA contents compared to those of the Florida population. MUFA content of muscle and liver membranes was higher in the Florida population compared to the Maine population. The effect of acclimation on CCO and CS activity was tissue-specific. In muscle, CCO and CS activities were higher in cold compared to warm-acclimated newts in both populations, and CS and CCO activities were higher in the Maine compared to the Florida population. In liver, CCO and CS activity were unaffected by acclimation in the Florida population, but activity was lower in cold compared to warm-acclimated Maine newts. These results demonstrate that the phenotypic plasticity of these traits in response to seasonal change has diverged between northern and southern populations.

Sexual dimorphism in a top predator (Notophthalmus viridescens) drives aquatic prey community assembly.

Intraspecific variation can have important consequences for the structure and function of ecological communities, and serves to link community ecology to evolutionary processes. Differences between the sexes are an overwhelmingly common form of intraspecific variation, but its community-level consequences have never been experimentally investigated. Here, we manipulate the sex ratio of a sexually dimorphic predacious newt in aquatic mesocosms, then track their impact on prey communities. Female and male newts preferentially forage in the benthic and pelagic zones, respectively, causing corresponding reductions in prey abundances in those habitats. Sex ratio differences also explained a large proportion (33%) of differences in the composition of entire pond communities. Ultimately, we demonstrate the impact of known patterns of sexual dimorphism in a predator on its prey, uncovering overlooked links between evolutionary adaptation and the structure of contemporary communities. Given the extreme prevalence of sexual dimorphism, we argue that the independent evolution of the sexes will often have important consequences for ecological communities.

The association between male-biased sex ratio and indicators of stress in red-spotted newts.

In populations with a male-biased operational sex ratio, coercive mating by males can have fitness consequences for females. One component of reduced fitness for females in populations with a male-biased OSR may be greater activation of the stress response, resulting in higher corticosterone release rates (CORT; a glucocorticoid stress hormone in amphibians). We test the hypothesis that a male-biased sex ratio affects female activity and release rates of CORT and testosterone (T) in male and female red-spotted newts (Notophthalmus viridescens). First, we evaluated if chemical cues from a male-biased sex ratio affect activity and CORT release rates in females. We predicted that females exposed to chemical cues of three males would be less active and have higher CORT release rates than those exposed to chemical cues of one male. Second, we measured CORT release rates of red-spotted newts in field enclosures with either a male-biased or a female-biased sex ratio. We predicted that females in the male-biased treatment would have higher CORT and T release rates than those in a female-biased treatment, owing to higher levels of male harassment. We also predicted that males would have higher CORT and T release rates in male-biased treatments due to higher levels of male-male competition. Females were not less active in response to chemical cues from more males over fewer males, but there was a positive relationship between female activity and CORT when they were exposed to the cues of three males. We also found that females, but not males, in the male-biased sex ratio treatment had higher CORT and T release rates than those in the female-biased treatment. Our results support the hypothesis that a male-biased sex ratio leads to a higher stress response, which may underlie the observed decrease in immune function and body condition in previous work exposing female red-spotted newts to a male-biased sex ratio. This study furthers our understanding of the mechanistic basis for costs associated with a male-biased sex ratio in a pond-breeding amphibian.

Comparative and developmental patterns of amphibious auditory function in salamanders.

Early amphibious tetrapods may have detected aquatic sound pressure using sound-induced lung vibrations, but their lack of tympanic middle ears would have restricted aerial sensitivity. Sharing these characteristics, salamanders could be models for the carryover of auditory function across an aquatic-terrestrial boundary without tympanic middle ears. We measured amphibious auditory evoked potential audiograms in five phylogenetically and ecologically distinct salamanders (Amphiuma means, Notophthalmus viridescens, Ambystoma talpoideum, Eurycea spp., and Plethodon glutinosus) and tested whether metamorphosis and terrestrial niche were linked to aerial sensitivity. Threshold differences between media varied between species. A. means' relative aerial sensitivity was greatest at 100 Hz and decreased with increasing frequency. In contrast, all other salamanders retained greater sensitivity up to 500 Hz, and in A. talpoideum and Eurycea, relative sensitivity at 500 Hz was higher than at 100 Hz. Aerial thresholds of terrestrial P. glutinosus above 200 Hz were similar to A. talpoideum and Eurycea, but lower than N. viridescens and A. means. Metamorphosis did not affect aerial sensitivity in N. viridescens or A. talpoideum. These results fail to support a hypothesis of terrestrial hearing specialization across ontogeny or phylogeny. We discuss methodological limitations to our amphibious comparisons and factors affecting variation in amphibious performance.

MARCKS-like protein is an initiating molecule in axolotl appendage regeneration.

Identifying key molecules that launch regeneration has been a long-sought goal. Multiple regenerative animals show an initial wound-associated proliferative response that transits into sustained proliferation if a considerable portion of the body part has been removed. In the axolotl, appendage amputation initiates a round of wound-associated cell cycle induction followed by continued proliferation that is dependent on nerve-derived signals. A wound-associated molecule that triggers the initial proliferative response to launch regeneration has remained obscure. Here, using an expression cloning strategy followed by in vivo gain- and loss-of-function assays, we identified axolotl MARCKS-like protein (MLP) as an extracellularly released factor that induces the initial cell cycle response during axolotl appendage regeneration. The identification of a regeneration-initiating molecule opens the possibility of understanding how to elicit regeneration in other animals.

Ecological Character Displacement between the Sexes.

Theory suggests that the evolution of sexual dimorphism in ecologically relevant traits can evolve purely through competition between the sexes for a shared resource. Although more parsimonious hypotheses exist for the evolution of ecological sexual dimorphisms, there are some underappreciated reasons to expect that competition may often play some role in the evolution of sexual dimorphism. Here, we build on past work to outline a set of sufficient criteria to demonstrate a role for resource competition in the evolution of sexual dimorphism, the most critical of which is that resource competition can be directly linked to sexual divergence along the axis of ecologically relevant dimorphism. We then compare the geometry of fitness surfaces across experimental manipulations of density and sex ratio in a semiaquatic salamander (Notophthalmus viridescens). We find consistent disruptive selection on multivariate sexual dimorphism in feeding morphology, which increases in strength with density. Fitness and the strength of divergent selection are negative-frequency dependent in the manner expected under competition-driven divergence between the sexes. Our results constitute direct evidence of resource competition as a driver of sexually antagonist selection and consequently the evolution of sexual dimorphism, providing an illustration of how cause and effect can be separated in studies of sexual divergence in morphology and ecology. We suggest that resource competition may often contribute to sexual divergence jointly with other sources of sex-biased selection, especially when ecological opportunity is sex specific.

Turning terminally differentiated skeletal muscle cells into regenerative progenitors.

The ability to repeatedly regenerate limbs during the entire lifespan of an animal is restricted to certain salamander species among vertebrates. This ability involves dedifferentiation of post-mitotic cells into progenitors that in turn form new structures. A long-term enigma has been how injury leads to dedifferentiation. Here we show that skeletal muscle dedifferentiation during newt limb regeneration depends on a programmed cell death response by myofibres. We find that programmed cell death-induced muscle fragmentation produces a population of 'undead' intermediate cells, which have the capacity to resume proliferation and contribute to muscle regeneration. We demonstrate the derivation of proliferating progeny from differentiated, multinucleated muscle cells by first inducing and subsequently intercepting a programmed cell death response. We conclude that cell survival may be manifested by the production of a dedifferentiated cell with broader potential and that the diversion of a programmed cell death response is an instrument to achieve dedifferentiation.

High pheromone diversity in the male cheek gland of the red-spotted newt Notophthalmus viridescens (Salamandridae).

BACKGROUND: Male salamanders (Urodela) often make use of pheromones that are produced in sexually dimorphic glands to persuade the female into courtship and mating. The mental gland of lungless salamanders (Plethodontidae) and dorsal cloacal glands (or abdominal glands) of newts (Salamandridae) have been particularly well studied in that respect. In both families, sodefrin precursor-like factor (SPF) proteins have been identified as major components of the courtship pheromone system. However, similar to plethodontids, some newts also make use of subtle head glands during courtship, but few pheromones have been characterized from such structures. Males of red-spotted newts (Notophthalmus viridescens, Salamandridae) have both cloacal and cheek (genial) glands, and are known to apply secretions to the female's nose by both tail-fanning and cheek-rubbing. Here we combined transcriptomic and phylogenetic analyses to investigate the presence, diversity and evolution of SPF proteins in the cloacal and cheek glands of this species. RESULTS: Our analyses indicate that the cheek glands of male N. viridescens produce a similar amount and diversity of SPF isoforms as the cloacal glands in this species. Expression in other tissues was much lower, suggesting that both male-specific courtship glands secrete SPF pheromones during courtship. Our phylogenetic analyses show that N. viridescens expresses a combination of isoforms that stem from four highly diverged evolutionary lineages of SPF variants, that together form a basis for the broad diversity of SPF precursors in the breeding glands. CONCLUSIONS: The similar SPF expression of cheek and cloacal glands suggests that this protein family is used for pheromone signalling through cheek rubbing in the red-spotted newt. Since several male salamandrids in other genera have comparable head glands, SPF application via other glands than the cloacal glands may be more widespread than currently appreciated in salamandrids.

Maintaining Eastern newts (Notophthalmus viridescens) for regeneration research.

The adult Eastern newt, Notophthalmus viridescens, has long served as a model for appendage as well as heart muscle regeneration studies. Newt tissues include all major cell types known in other vertebrates and mammals, including bone, cartilage, tendon, muscle, nerves, dermis, and epidermis. Therefore, these aquatic salamanders make an excellent model for studying the regeneration of complex tissues. Regeneration of adult tissues requires the integration of new tissues with preexisting tissues to form a functioning unit through a process that is not yet well understood. Scale is also an issue, because the regenerating tissues or structures are magnitudes larger than their embryonic counterparts during development, and therefore, it is likely that different physics and mechanics apply. Regardless, regeneration recapitulates to some degree developmental processes. In this chapter, we will describe basic methods for maintaining adult Eastern newts in the laboratory for the study of regeneration. To determine similarities and differences between development and regeneration at the cellular and molecular level, there is also a need for embryonic newt tissue. We therefore also outline a relatively simple way to produce and raise newt embryos in the laboratory.

Studying newt brain regeneration following subtype specific neuronal ablation.

The realization that neuronal injury does not result in permanent functional or cellular loss in all vertebrates has fascinated regenerative biologists. Neuronal regeneration occurs in a subset of species, including lizards, teleost fish, axolotls, and newts. One tool for studying neuronal regeneration in the adult brain is intraventricular injection of selective neuronal toxins, which leads to loss of subpopulations of neurons. To trace cells involved in the regeneration process, plasmids encoding reporter proteins can be electroporated in vivo into the cells of interest. This protocol describes methods to label the ependymoglial cells of the brain of the red spotted newt Notophthalmus viridescens and follow their response after ablation of dopaminergic neurons.

Does the thermal plasticity of metabolic enzymes underlie thermal compensation of locomotor performance in the eastern newt (Notophthalmus viridescens)?

Eastern newts (Notophthalmus viridescens) upregulate the metabolic capacity of skeletal muscle in winter to compensate for thermodynamic effects on metabolism. However, whether this compensation facilitates locomotor performance at low temperature is unknown. Therefore, our aim was to determine if thermal acclimation of metabolic enzymes in muscle benefits locomotion. Eastern newts from southern Ohio were acclimated to cold (5°C, 10:14 L:D) or warm (25°C, 14:10 L:D) conditions for 12 weeks. Following acclimation, we measured the locomotor performance (burst speed and time until exhaustion) and the activities of metabolic enzymes in skeletal muscle at 5-30°C. Creatine kinase (CK) activity in skeletal muscle was higher in cold compared to warm-acclimated newts, and cold-acclimated newts had a higher burst speed at low temperature compared to warm-acclimated newts. At low temperature, time until exhaustion was higher in cold compared to warm-acclimated newts, but the activities of citrate synthase (CS) and cytochrome c oxidase (CCO) in muscle were lower in cold compared to warm-acclimated newts. Together, these results demonstrate that eastern newts compensate for the effects of low temperature on locomotor performance. Whereas thermal compensation of CK activity is correlated with burst locomotion at low temperature, aerobic enzymes in skeletal muscle (CS and CCO) are not linked to compensation of sustained locomotion.

An acute increase in the stress hormone corticosterone is associated with mating behavior in both male and female red-spotted newts, Notophthalmus viridescens.

Hormones play key, functional roles in mediating the tradeoff between survival and reproduction. Glucocorticoid hormones can inhibit reproduction and improve chances of survival during periods of stress. However, glucocorticoid hormones are, at times, also associated with successfully engaging in energetically costly courtship and mating behaviors. Corticosterone (CORT), a primary glucocorticoid hormone in amphibians, reptiles and birds, may be important in activating or sustaining energetically costly mating behaviors. We used a non-invasive, water-borne hormone assay to measure CORT release rates of male and female red-spotted newts (Notophthalmus viridescens) collected when either engaged in amplexus or when not engaged in amplexus. Because amplexus is energetically costly for males, we predicted that males would have higher CORT release rates than females. We also predicted that females in amplexus would have elevated CORT release rates because the restraint of amplexus prevents foraging and breathing and may be costly. Here we show that an acute increase in CORT is associated with amplexus behavior in both male and female red-spotted newts. Additionally we demonstrate that males have higher overall CORT release rates both in and out of amplexus than do females. Our results support the hypothesis that glucocorticoid hormones are associated with energetically costly courtship and mating behaviors for both sexes.

Interactive effects of competition and social environment on the expression of sexual dimorphism.

The expression of sexual dimorphism is expected to be influenced by the acquisition of resources available to allocate to trait growth, combined with sex-specific patterns of resource allocation. Resource acquisition in the wild may be mediated by a variety of ecological factors, such as the density of interspecific competitors. Allocation may in turn depend on social contexts, such as sex ratio, that alter the pay-off for investment in sexual traits. How these factors interact to promote or constrain the expression and evolution of sexual dimorphism is poorly understood. We manipulated sex ratio and interspecific resource competition over the growing season of red-spotted newts (Notophthalmus viridescens) in artificial ponds. Fish competitors had a stronger effect on female than male growth, which effectively eliminated the expression of sexual size dimorphism. In addition, newt sex ratio influenced fish growth, leading to reduction in fish mass with an increase in female newt frequency. Fish also reduced the expression of male tail height, a sexually selected trait, but only in tanks with a female-biased sex ratio. This suggests males alter their resource allocation pattern in response to the strength of sexual selection. Our results demonstrate that ecologically and socially mediated interactions between sex-specific resource acquisition and allocation can contribute to variation in the expression of sexual dimorphism.

A focus on the optical properties of the regenerated newt lens.

Lens regeneration studies in the adult newt suggest that molecular aspects of lens regeneration are complete within 5 weeks of lentectomy. However, very little is known about the optical properties of the regenerated lens. In an aquatic environment, the lens accounts for almost all of the refractive power of the eye, and thus, a fully functional lens is critical. We compared the optical properties of 9- and 26-week regenerated lenses in the red spotted newt, Notophthalmus viridescens, with the original lenses removed from the same eyes. At 9 weeks, the regenerated lenses are smaller than the original lenses and are histologically immature, with a lower density of lens proteins. The 9 week lenses have greater light transmission, but significantly reduced focal length and refractive index than the original lenses. This suggests that following 9 weeks of regeneration, the lenses have not recovered the functionality of the original lens. By 26 weeks, the transmission of light in the more mature lens is reduced, but the optical parameters of the lens have recovered enough to allow functional vision.

Transcriptome analysis of newt lens regeneration reveals distinct gradients in gene expression patterns.

Regeneration of the lens in newts is quite a unique process. The lens is removed in its entirety and regeneration ensues from the pigment epithelial cells of the dorsal iris via transdifferentiation. The same type of cells from the ventral iris are not capable of regenerating a lens. It is, thus, expected that differences between dorsal and ventral iris during the process of regeneration might provide important clues pertaining to the mechanism of regeneration. In this paper, we employed next generation RNA-seq to determine gene expression patterns during lens regeneration in Notophthalmus viridescens. The expression of more than 38,000 transcripts was compared between dorsal and ventral iris. Although very few genes were found to be dorsal- or ventral-specific, certain groups of genes were up-regulated specifically in the dorsal iris. These genes are involved in cell cycle, gene regulation, cytoskeleton and immune response. In addition, the expression of six highly regulated genes, TBX5, FGF10, UNC5B, VAX2, NR2F5, and NTN1, was verified using qRT-PCR. These graded gene expression patterns provide insight into the mechanism of lens regeneration, the markers that are specific to dorsal or ventral iris, and layout a map for future studies in the field.

A reference transcriptome and inferred proteome for the salamander Notophthalmus viridescens.

Salamanders have a remarkable capacity to regenerate complex tissues, such as limbs and brain, and are therefore an important comparative model system for regenerative medicine. Despite these unique properties among adult vertebrates, the genomic information for amphibians in general, and salamanders in particular, is scarce. Here, we used massive parallel sequencing to reconstruct a de novo reference transcriptome of the red spotted newt (Notophthalmus viridescens) containing 118,893 transcripts with a N50 length of 2016 nts. Comparisons to other vertebrates revealed a newt transcriptome that is comparable in size and characteristics to well-annotated vertebrate transcriptomes. Identification of putative open reading frames (ORFs) enabled us to infer a comprehensive proteome, including the annotation of 19,903 newt proteins. We used the identified domain architectures (DAs) to assign ORFs phylogenetic positions, which also revealed putative salamander specific proteins. The reference transcriptome and inferred proteome of the red spotted newt will facilitate the use of systematic genomic technologies for regeneration studies in salamanders and enable evolutionary analyses of vertebrate regeneration at the molecular level.

A microarray analysis of gene expression patterns during early phases of newt lens regeneration.

PURPOSE: Notophthalmus viridescens, the red-spotted newt, possesses tremendous regenerative capabilities. Among the tissues and organs newts can regenerate, the lens is regenerated via transdifferentiation of the pigment epithelial cells of the dorsal iris, following complete removal (lentectomy). Under normal conditions, the same cells from the ventral iris are not capable of regenerating. This study aims to further understand the initial signals of lens regeneration. METHODS: We performed microarray analysis using RNA from a dorsal or ventral iris isolated 1, 3, and 5 days after lentectomy and compared to RNA isolated from an intact iris. This analysis was supported with quantitative real-time polymerase chain reaction (qRT-PCR) of selected genes. RESULTS: Microarrays showed 804 spots were differentially regulated 1, 3, and 5 days post-lentectomy in the dorsal and ventral iris. Functional annotation using Gene Ontology revealed interesting terms. Among them, factors related to cell cycle and DNA repair were mostly upregulated, in the microarray, 3 and 5 days post-lentectomy. qRT-PCR for rad1 and vascular endothelial growth factor receptor 1 showed upregulation for the dorsal iris 3 and 5 days post- lentectomy and for the ventral iris 5 days post-lentectomy. Rad1 was also upregulated twofold more in the dorsal iris than in the ventral iris 5 days post-lentectomy (p<0.001). Factors related to redox homeostasis were mostly upregulated in the microarray in all time points and samples. qRT-PCR for glutathione peroxidase 1 also showed upregulation in all time points for the ventral and dorsal iris. For the most part, mitochondrial enzymes were downregulated with the notable exception of cytochrome c-related oxidases that were mostly upregulated at all time points. qRT-PCR for cytochrome c oxidase subunit 2 showed upregulation especially 3 days post-lentectomy for the dorsal and ventral iris (p<0.001). Factors related to extracellular matrix and tissue remodeling showed mostly upregulation (except collagen I) for all time points and samples. qRT-PCR for stromelysin 1/2 alpha and avidin showed upregulation in all the time points for the dorsal and ventral iris. CONCLUSIONS: The results show that the dorsal iris and the ventral iris follow the same general pattern with some distinct differences especially 5 days after lentectomy. In addition, while the expression of genes involved in DNA repair, redox homeostasis, and tissue remodeling in preparation for proliferation and transdifferentiation is altered in the entire iris, the response is more prominent in the dorsal iris following lentectomy.

Temperature preference during forelimb regeneration in the red-spotted newt Notophthalmus viridescens.

Red-spotted newts (Notophthalmus viridescens) are model organisms for regenerative research. These animals can regenerate limbs, tails, jaws, spinal cords, as well as the lens of the eye. Newts are small ectotherms that are aquatic as adults; as ectotherms, they naturally conform to the temperature of their surroundings. Environmental temperatures, however, can increase or decrease the red-spotted newt's metabolic processes, including their rate of tissue regeneration; whether an optimal temperature for this rate of regeneration exists is unknown. However, newts do exhibit behavioral preferences for certain temperatures, and these thermal preferences can change with season or with acclimation. Given this flexibility in behavioral thermoregulation, we hypothesized that the process of tissue regeneration could also affect thermal preference, given the metabolic costs or altered temperature sensitivities of tissue regrowth. It was predicted that regenerating newts would select an environmental temperature that maximized the rate of regeneration, however, this prediction was not fully supported. Thermal preference trials revealed that newts consistently selected temperatures between 24 and 25°C throughout regeneration. This temperature selection was warmer than that of uninjured conspecifics, but was lower than temperatures that would have further augmented the rate of regeneration. Interestingly, regenerating newts maintained a more stable temperature preference than sham newts, suggesting that accuracy in thermoregulation may be more important to regenerating individuals, than to noninjured individuals.